1. Field of the Invention
This invention relates to microporous membrane modified by or having bonded thereto ionene polymer and the use of the polyionene-transformed microporous membrane for the separation of contaminants of microorganism origin from biological liquids.
2. Brief Description of the Background Art
Removal of microorganism-originated contaminants from biological liquids has long been a recalcitrant problem. Examination of bacterial cell wall surface properties has demonstrated that both gram-positive and gram-negative bacteria are negatively charged, mainly due to an excess of carboxyl and phosphate groups in the cell walls thereof. Gram-positive bacteria contain both teichoic and teichuronic acids in their cell walls, whereas gram-negative organisms have phospholipids and the negatively lipid A portion of lipopolysaccharides as components of their outer cell membranes. In aqueous environments, the cell membrane exists as a continuum of lipid and protein organized as a molecular double layer, with the hydrophobic portions of the lipid molecules being opposed and the hydrophilic group projecting outwardly into the aqueous phase. Phosphoglycerides account for about half the lipid with polar groups, such as glycerol, serine, and carboxyl, providing the hydrophilic components. While various protein forms are imbedded in the lipid, the major determinants of charge are surface polysaccharides covalently linked to the membrane protein and lipids.
The effectiveness of bacteria removal through charge interaction has been previously demonstrated, for example, by Ostreicher et al., U.S. Pat. No. 4,305,782. Capture of bacteria, endotoxins, and viruses by charge modified filters are described in Applied and Environmental Microbiology, 40: 892-896 (1980). Positively charged ion exchange resins have been utilized for bacteria adsorption (Daniels, S. L., Development and Industrial Microbiology, 13: 211-253 (1972)).
Olson et al., U.S. Pat. No. 4,411,795 describes a variety of polymers attached to substrates including nylon and recognized that the combined effect of hydrophilic and ionic binding enhances adsorption of lipin-containing cells.
Zvaginstev, D. G. et al., Mikrobiologiya 40: 123-126 (1971), concluded that adsorption of bacterial cells by ion exchange resins was attributable to electrostatic attraction between quaternary ammonium groups on the resin surface and carboxyl groups on the bacteria cell surface. Hogg, in his Ph.D. thesis for the University of Salford, England (1976), demonstrated the interaction of bacteria with cellulose-based DEAE. The adsorption of several gram-negative organisms was shown, including Escherichia coli, Salmonella typhimurium, and Pseudomonas aeruginosa.
The major forces impacting on bacterial adhesion to solid surfaces have been summarized by Rutter, P. R. in "The Physical Chemistry of the Adhesion of Bacteria and Other Cells," Microbial Adhesion to Surfaces, Editors Berkley et al., Ellis Howard Ltd. Publishers, West Sussex, England (1980). According to Rutter, the Van der Walls force and charge interaction may be considered as long range forces. Where the distance between bacteria and solid surfaces are short, other interactions must be taken into account, for example, ion-dipole, dipole-dipole, hydrogen bonding, etc. The short range effects are particularly important in aqueous systems. When the bacteria particles approach the microscopic solid surface, the local ordered water structure near the surface must be broken down. This leads to a short range repulsion force, which may be sufficient to prevent the bacteria from coming closer to the solid surface. On the other hand, when both the surfaces involved are hydrophobic, the short range interaction is a net attraction. This energy favorable process, called "hydrophobic interaction," is the basis for the well-known high performance liquid chromatography applied in protein separations. As is known, a hydrocarbon chain of optimum length, when attached covalently to a solid matrix, may adsorb one protein in preference to another due to the difference in hydrophobicity between proteins.
However, an overly strong hydrophobic solid surface may uncoil the protein structure leading to the exposure of hydrophobic regions and increase tendency for hydrophobic interaction. If the uncoiling is too extensive, denaturation of the protein may result.
In most of the practical applications for bacteria and endotoxin inactivation and removal from biological and pharmaceutical products, protein contamination by bacteria is the most prevalent problem. One must be able to inactivate and remove the microorganism-originated contaminants from protein specifically without causing loss or denaturation of the final products. Accordingly, an optimal solid matrix should exhibit a hydrophobic force which just matches the surface hydrophobicity of proteins and maximally exploits these selected differences.
Thus, a need has continued to exist for a solid matrix for removal of microorganism-originated contaminants from biological and pharmaceutical products which will effectively eliminate the contaminants without denaturing the final product.
Microporous membranes and their use for sterile filtration are well-known in the art. U.S. Pat. No. 3,876,738 to Marinaccio et al. describes a process for preparing a microporous membrane. U.S. Pat. No. 4,340,479 to Pall describes a similar process. Additional processes for producing microporous membranes are described in U.S. Pat. No. 3,642,668 to Bailey et al.; U.S. Pat. No. 4,203,847 to Grandine, II; U.S. Pat. No. 4,203,848 to Grandine, II; and U.S. Pat. No. 4,247,498 to Castro.
Commercially available microporous membranes, for example, made of nylon, are available from Pall Corporation, Glen Cove, N.Y., under the trademark ULTIPORE N.sub.66.sup.R. Another commercially significant membrane made of polyvinylidene fluoride is available from Millipore Corp., Bedford, Mass., under the trademark DURAPORE.RTM.. Each of the above membranes is advertised as useful for filtration of pharmaceuticals for removal of microorganisms and the like.
Efforts to improve the bacterial retention of various microporous membranes has resulted in charge modification of various microporous membranes. Assignee's U.S. Pat. No. 4,473,474 to Ostreicher et al. describes a particularly preferred charge-modified microporous membrane wherein the membrane, preferably nylon, comprises a multiplicity of cationic charge sites on the internal pore surfaces. The charge sites are provided by a cationic charge-modifying resin, in particular a polyamido-polyamine epichlorohydrin resin, bonded to the membrane structure. The membrane may be further provided with a cross-linking agent for the charge modifying resin which is effective in retaining the resin on the membrane. This membrane is sold under the trademark ZETAPOR.RTM. by AMF Cuno, Meriden, Conn. A substantial advantage to the charge-modified microporous membranes known to the prior art resides in the action of exclusion of particulate on the basis of charge as well as size. Thus, for example, viruses may be removed from fluid without having to go to an ultrafiltration membrane with its associated high pressures. One problem encountered by such prior art membranes, specifically the aforementioned ZETAPOR.RTM. cationically modified nylon membrane is the extremely slow flush out characteristic which prevents the use of such charoe-modified media for certain applications.
As an improvement upon the ZETAPOR.RTM. membrane described above, assignee obtained U.S. Pat. No. 4,473,475 to Barnes, Jr. et al., a patent disclosing a cationic charge-modified microporous membrane. The membrane comprises a hydrophilic organic polymeric microporous membrane having bonded thereto, through a cross-linking agent, a charge modifying amount of a cationic charge-modifying agent. The charge-modifying agent is an aliphatic amine or polyamine; the cross-linking agent is an aliphatic polyepoxide; the preferred microporous membrane is nylon.
Hou et al., U.S. Pat. No. 4,361,486, discloses a bactericidal filtration media which comprises an amount of metal peroxide immobilized in a substantially inert porous matrix.
However, in spite of continued efforts to improve the separation characteristics of such microporous membranes for the removal of microorganism-originated contaminants from biological liquids, "bacterial breakthrough" remains an enduring problem. One explanation for this phenomenon resides in the fact that bacteria, although retained on the separation membrane, continue to grow and multiply thereon. Subsequently, when the bacterial population becomes of sufficient size, bacterial contamination of the biological liquids flowing therethrough results. Thus, a need has continued to exist for microporous membrane having the capability of high efficiency separation of microorganism-originated contaminants from biological liquids.
Additionally, ion rejecting membranes are known. U.S. Pat. No. 4,125,462 to Latty describes a coated membrane having a layer or coating of a cationic polyelectrolyte, preferably poly(vinylimidazoline) in the bisulfate form. Other types of charge-modified membranes are described in U.S. Pat. No. 3,556,992 to Massucco; U.S. Pat. No. 3,556,305 to Shorr; U.S. Pat. No. 3,808,305 to Gregor; and U.S. Pat. No. 4,250,029 to Kisset et al.
Poly-quaternary ammonium polymeric polyelectrolytes are known to the prior art; these polymeric compositions are produced by the polymerization of a dihalide and a ditertiary amine. These polymers are characterized by high charge density and have found substantial utility as flocculants in the clarification of residential and industrial water supplies, as catalysts in pigment retention additives, and as geling agents. These polyelectrolyte materials are also known to be useful in the rheological modification of fluids such as friction reducers, as dispersants for clay and sludge in both aqueous and oil-based systems, as anti-static agents, and as additives to cosmetics, textile finishes and lubricating oils. The materials are known to exhibit germicidal action or effective bactericidal and fungicidal agents. See Rembaum et al., U.S. Pat. No. 3,898,188.
Buckman et al., U.S. Pat. No. 3,784,649, discloses "high molecular weight" ionene polymeric compositions for utility, among others, as broad spectrum microbicides for efficient control of bacteria including sulphate reducers, fungi, algae, and yeast. The Buckman et al. polyionenes are suggested as additives to paper making systems, the polyionenes increasing production per unit of equipment, improving formation and strength properties of paper and paper board, and alleviating water pollution problems.
Rembaum, U.S. Pat. No. 4,046,750, discloses ionene modified beads for use in binding small and large anionic compounds. The bead substrates are formed by the aqueous copolymerization of a substituted acrylic monomer and a cross-linking agent. The formed polymeric beads are reacted with a mixture of a ditertiary amine and a dihalide or with a dimethylaminoalkyl halide to attach ionene segments to the halo or tertiary amine centers on the beads. The thus-formed polyionene-modified beads find use in affinity or pellicular chromatography for removal of heparin from its mixture with polycations or neutral substances such as proteins or serums. Further disclosed utilities include use of the modified beads in the separation of cholesterol precursors such as bile acid from bile micellar suspensions, for binding RNA or DNA irreversibly, and a variety of other utilities which depend upon the binding characteristics of the polycationic nature of the polyionene.
Rembaum, U.S. Pat. No. 4,013,507, discloses ionene polymers which bind negatively charged mammalian cells such as malignant cells for selectively inhibiting the growth in vitro thereof. Conversely, U.S. Pat. No. 3,910,819 to Rembaum et al. discloses the use of polyionene-coated containers for increasing the rate of cell growth.
U.S. Pat. No. 3,927,242 to Rembaum et al. discloses the use of polyionenes as coatings for paper substrates. Further disclosed are substrates coated with the polyelectrolyte to maximize the bactericidal activity of the polyionene. Suggested utilities include the impregnation of gauze material to form an antiseptic coagulant, germicidal dressing material.
U.S. Pat. No. 4,075,136 to Schaper discloses a class of ionene polymers which contain certain functional groups such as nitriles, acrylates, vinyl acetates, ketones, acrolein, acrylamides, methosulfates, sulfonic acids, pyridines, and pyrrolidones. A host of utilities are disclosed, including the use of the functional ionene polymers as biocides and as functional coatings on paper, for example, electroconductive, adhesive and photosensitive coatings.
However, prior to the present invention, the use of polyionenes to transform and improve the capture and inactivation of contaminants of microorganism origin has not been appreciated.